Thirst interneurons that promote water seeking and limit feeding behavior in Drosophila - eLife
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Thirst interneurons that promote water
seeking and limit feeding behavior in
Drosophila
Dan Landayan1†, Brian P Wang1, Jennifer Zhou2, Fred W Wolf1,2*
1
Quantitative and Systems Biology Graduate Program, UC, Merced, United States;
2
Department of Molecular and Cell Biology, UC, Merced, United States
Abstract Thirst is a motivational state that drives behaviors to obtain water for fluid
homeostasis. We identified two types of central brain interneurons that regulate thirsty water
seeking in Drosophila, that we term the Janu neurons. Janu-GABA, a local interneuron in the
subesophageal zone, is activated by water deprivation and is specific to thirsty seeking. Janu-AstA
projects from the subesophageal zone to the superior medial protocerebrum, a higher order
processing area. Janu-AstA signals with the neuropeptide Allatostatin A to promote water seeking
and to inhibit feeding behavior. NPF (Drosophila NPY) neurons are postsynaptic to Janu-AstA for
water seeking and feeding through the AstA-R2 galanin-like receptor. NPF neurons use NPF to
regulate thirst and hunger behaviors. Flies choose Janu neuron activation, suggesting that thirsty
seeking up a humidity gradient is rewarding. These findings identify novel central brain circuit
elements that coordinate internal state drives to selectively control motivated seeking behavior.
*For correspondence: Introduction
fwolf@ucmerced.edu Thirst is the neural representation of the internal state caused by dehydration or decreased blood
† volume. Thirst motivates a coordinated series of water seeking and intake behaviors across diverse
Present address: Department
animal phyla. Terrestrial animals with unpredictable access to patchily distributed sources of water,
of Psychiatry and Behavioral
Sciences, Stanford University, such as the fly Drosophila melanogaster, have evolved robust brain circuitry to rapidly detect water
Stanford, United States loss to drive goal-directed seeking and ingestion behaviors.
In flies, water ingestion and humidity preference studies led to the discovery of sensory and cen-
Competing interests: The
tral brain circuit elements that control behavioral and hormonal aspects of thirst. Flies rapidly detect
authors declare that no
humidity gradients via humid-sensing (Ir68a receptor) and dry-sensing (Ir40a receptor) hygrosensory
competing interests exist.
neurons that are housed in the sacculus on the antennae (Enjin et al., 2016; Ji and Zhu, 2015;
Funding: See page 16 Knecht et al., 2016; Knecht et al., 2017). The hygrosensory neurons project to dedicated glomeruli
Received: 08 January 2021 in the antennal lobe, where they synapse onto second order projection neurons to send information
Accepted: 30 April 2021 to the mushroom bodies, lateral horn, and broadly to other areas of the protocerebrum
(Frank et al., 2017; Marin et al., 2020). Water taste is facilitated by water-sensing (PPK28 mechano-
Reviewing editor: Mani
Ramaswami, Trinity College
receptor) gustatory neurons; taste is critical for water consumption in thirsty flies (Cameron et al.,
Dublin, Ireland 2010; Lau et al., 2017). A pair of interoceptive SEZ neurons (ISN) that detect osmolarity are located
in the suboesophageal ganglion zone (SEZ); they promote water consumption and reciprocally
Copyright Landayan et al. This
inhibit food intake when inhibited by high interstitial fluid osmolarity (Jourjine et al., 2016). The
article is distributed under the
ISNs are regulated by the mechanoreceptor Nanchung that responds to changes in hemolymph
terms of the Creative Commons
Attribution License, which osmolarity to signal water balance status, and by adipokinetic hormone that signals aspects of
permits unrestricted use and energy store status to the brain.
redistribution provided that the These sensory and interoceptive inputs regulate central brain circuitry to facilitate ingestive
original author and source are behavior and initiate the state of thirst. How the thirsty state is represented in the brain remains to
credited. be characterized. Functional mapping of water reward memories led to the discovery of leucokinin
Landayan et al. eLife 2021;10:e66286. DOI: https://doi.org/10.7554/eLife.66286 1 of 19
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peptidergic neurons, whose activity is regulated by thirst, and that regulate memories of thirst
through dopaminergic connections into the mushroom body learning and memory circuitry (Ji and
Zhu, 2015; Lin et al., 2014; Senapati et al., 2019). Thirsty attraction to humidity may also route
through some of the same dopamine neurons (Lin et al., 2014). Hunger, which drives a pattern of
ingestive behaviors similar to that of thirst, is also conveyed through dopamine neurons and the
mushroom body circuitry (Tsao et al., 2018). However, the role of hunger circuitry in water seeking
is not yet known. Finally, the pars intercerebralis, a neuroendocrine brain region, contains ion trans-
port peptide (ITP) hormone expressing neurons (Gáliková et al., 2018; Nässel and Vanden Broeck,
2016). Ion transport peptide bidirectionally controls thirst and hunger behaviors, and it regulates
water balance in the body as a hormone.
Understanding how fundamental motivational states are related at the circuit level will help
uncover the hierarchy of behavioral choice and its underlying neural mechanisms. Thirst in mammals
has complex hierarchical interactions with other behavioral states, including hunger and sleep
(Sutton and Krashes, 2020). Thirst and hunger are particularly tightly interconnected. For example,
thirst can override hunger drive (dehydration-anorexia), and feeding induces thirst (prandial thirst)
(Watts and Boyle, 2010). Manipulation of either cortical or hindbrain neurons in mice can simulta-
neously impact both hunger and thirst driven ingestive behavior (Eiselt et al., 2021; Gong et al.,
2020). Moreover, thirst-driven water anticipation and subsequent intake changes the activity of a
remarkably broad set of neurons throughout the mouse brain, in a manner that is time-locked to
specific behavioral steps (Allen et al., 2019). This reveals the complexity of even an apparently sim-
ple motivational state, and it predicts interactions with other brain functions. Flies, exhibiting hun-
ger:thirst and hunger:sleep interdependencies with apparently equivalent ethological relationships,
will be useful for elaborating circuit motifs for behavioral state interactions, particularly at the level
of individual neurons (Melnattur and Shaw, 2019).
To identify central brain neurons critical for thirst, we used an open-field behavioral assay that
allows neural manipulation of freely moving animals as they evaluate the presentation of a point
source of water or food. An unbiased neuroanatomical activation screen identified two bilateral pairs
of neurons, Janu-GABA and Janu-AstA, whose activity is necessary and sufficient for promoting
thirsty water seeking, but that have limited effect on fluid consumption. Flies choose to continuously
activate the Janu neurons, suggesting that water seeking up a humidity gradient is rewarding. The
Janu-GABA local interneurons are specific to thirsty water seeking. In contrast, the Janu-AstA neu-
rons, that release the neuropeptide Allatostatin A (AstA), also inhibit feeding behavior. Neuropep-
tide F (NPF) neurons are a postsynaptic target of Janu-AstA neurons through the AstA-R2 receptor
in the superior medial protocerebrum. NPF reciprocally regulates water seeking and feeding behav-
ior. Thus, we uncovered central brain interneurons that encode water seeking and that simulta-
neously suppress food seeking, indicating that thirst and hunger are coordinated homeostatic states
while animals are actively seeking.
Results
Anatomical activation screen identifies water-seeking neurons
Thirsty flies are motivated to seek a palatable source of water and to drink until they reach repletion
(Figure 1A). We developed an open-field assay to assess appetitive behaviors, including thirst
(Figure 1B). Flies are placed into an open arena, allowed to acclimate, and then presented with a
small receptacle containing a source of water or food. A water source rapidly sets up a stable 3–6%
humidity gradient from the edge to the source at the center of the chamber. Flies may perform a
complete series of steps of thirst-driven behavior with an accessible water source, from seeking to
achieving repletion, or they can be limited to water seeking by placing a mesh grid over the source.
The longer flies are water deprived, the more quickly they find and occupy the water source
(Figure 1C,D). Water deprivation also increases locomotor speed (Figure 1E). Given a choice, thirsty
flies seek water over food, and hungry flies seek food over water, indicating that deprivation state-
specific seeking can be readily measured in the open field assay (Figure 1F). A combination of
hygrosensory and taste cues guide thirsty flies to water. Blocking access to the water source with the
mesh grid had no effect on seeking, whereas removal of the third antennal segment that harbors
hygrosensory neurons in the sacculus (antennectomy) reduced seeking (Figure 1G). Combining
Landayan et al. eLife 2021;10:e66286. DOI: https://doi.org/10.7554/eLife.66286 2 of 19
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A B Replete Thirsty C H2O deprived D
search 2 hr 12 hr
1 1
Fraction on H2O
Fraction on H2O
** ** ** **
contact
*
0.5 0.5
taste
drink
0 0
2 4 8 12 16 20 24
disengage 0 5 10 15
H2O deprivation, hr
Time, min
E F Preference index, H2O G H
10 1 ** 1 4
**
Fraction on H2O
H2O intake, a.u.
* **
Speed, mm/sec
8 ** 3 **
6 **** **
0 0.5 2
4
2 ** 1
-1
**
0 0 0
0 2 4 6 8 12 16 Thirsty • • • • • Thirsty • • •
H sty
y
gr
H2O deprivation, hr Grid • • • Grid •
ir
un
Th
Antennectomy • • Antennectomy •
Aristae ablation •
I 1 J ** ** K **
attraction 1 1
Fraction on source
Fraction on H2O
Durstig
Seeking index, food
** **
0.5
0.5 0.5
0
0 0
Durstig-Gal4 • • • • • • Durstig-Gal4 • • •• ••
-0.5 aversion UAS-TrpA1 • • • • • • UAS-TrpA1 • • • • • •
wet H2O dry Ant – Ant –
sucrose sucrose Grid Grid
H2O replete, fed H2O replete, fed
Figure 1. Thirst behavior in an open field; a forward screen uncovers Durstig thirst neurons. (A) Thirst-induced sequence of behavior. (B) Two-
chambered open field assay with a vertical divider at the center. Thirsty flies avidly seek and occupy a discrete water source (dyed blue for clarity). (C)
Occupancy of an open water source over time by a group of 20 flies, n = 8 groups. (D) Occupancy increases with increasing water deprivation. One-way
ANOVA/Dunnett’s compared to 2 hr water deprivation. Each dot represents one group of 20 flies. (E) Water deprivation increases locomotor activity.
One-way ANOVA/Dunnett’s compared to 0 hr. (F) Two choice preference for water (1) and dry sucrose ( 1) depends on deprivation state. One-sample
t-test, compared to 0 (no preference). (G) Sensory systems in thirsty water seeking. A mesh grid atop the water source preserves humidity sensing and
blocks water contact, taste, and ingestion. Antennectomy, removal of the third antennal segment and aristae, removes humidity and temperature
sensors. One-way ANOVA/Dunnett’s compared to open/unoperated. (H) Sensory input in water intake. Kruskal-Wallis/Dunn’s compared to water
replete (gray bar). (I) Screen for neurons that affect the occupancy of food, using a library of InSITE enhancer-Gal4 strains driving expression of the
TrpA1 heat activated cation channel. Seeking index is enhancer-Gal4>TrpA1 minus enhancer-Gal4>+. Positive index indicates greater occupancy. Gray
extends to two standard deviations from the mean. (J) Durstig neuron activation drives occupancy of food and water. (K) Durstig activation promotes
water occupancy through hygrotaxis and contact-dependent mechanisms. Statistics are one-way ANOVA/Tukey’s or Kruskal-Wallis/Dunn’s, unless
indicated otherwise.
The online version of this article includes the following figure supplement(s) for figure 1:
Figure 1 continued on next page
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Figure 1 continued
Figure supplement 1. Effects of activating neurons implicated in feeding, reward, and water learning on water seeking.
antennectomy with the mesh grid completely blocked seeking. Antennectomy also removes the aris-
tae that sense temperature; ablation of the aristae alone had no effect on seeking to the inaccessible
water source (Budelli et al., 2019). Water deprivation increased water intake: removing the third
antennal segment had no effect, whereas, as expected, blocking access with the mesh grid fully
blocked intake (Figure 1H). Thirsty flies were not attracted to an empty receptacle (not shown).
Thus, thirsty flies use hygrosensory and taste environmental cues to locate and drink water, and we
can isolate the hygrotactic water seeking step from other thirst-related behaviors.
To identify neurons that promote seeking behavior, we acutely activated neurons in 154 different
InSITE enhancer-Gal4 patterns in water replete, well-fed (satiated) flies (Gohl et al., 2011). We ini-
tially screened for feeding behavior by presenting flies with a discrete source of standard fly food.
The screen revealed patterns of neurons that, when activated by the heat-sensitive TrpA1 cation
channel, promote or inhibit foraging (Figure 1I). One activation pattern, Durstig-Gal4 (the German
for ‘thirst’), resulted in markedly high attraction to food. We asked what component of the food
source the Durstig>TrpA1 flies were seeking. Well fed, water replete Durstig>TrpA1 flies avidly
occupied standard fly food, wet sucrose or yeast alone in 1% agar, dry sucrose, 1% agar alone, or
water alone, but they were not attracted to empty receptacles (Figure 1J). Hygrotaxis toward inac-
cessible water was strongly potentiated by Durstig activation (Figure 1K). Thus, Durstig contains
neurons that promote water seeking. We chose to focus on water seeking behavior because it is a
specific step in the thirst behavioral sequence that is strongly motivated.
We asked if neurons previously implicated in intake (food and water), water reward memory, and
water sensing increase water seeking when activated. Activation of dopamine neurons for reward
and aversion had either no effect or suppressed water seeking (Figure 1—figure supplement 1A).
Moreover, dopaminergic neurons previously implicated in humidity preference and water reward, in
the R48B04 pattern, are different from the Durstig water seeking neurons (Figure 1—figure supple-
ment 1B). Of neurons implicated in feeding, activation of only two patterns increased water seeking:
the NP883 pattern containing the Fdg neurons that promote proboscis extension and liquid food
intake, and the R65D05 pattern that contains AstA neurons that inhibit food intake (Figure 1—figure
supplement 1C; Chen et al., 2016; Flood et al., 2013; Hentze et al., 2015; Hergarden et al.,
2012; Pool et al., 2014). Activation of internal osmosensory ISNs (R34G02), hygrosensory neurons
(Ir40a, Ir68a), and water taste neurons (ppk28) did not increase water seeking (Figure 1—figure sup-
plement 1D; Enjin et al., 2016; Jourjine et al., 2016; Knecht et al., 2016). These findings reveal
segregation of water seeking from many of the previously identified neurons involved in steps of
feeding and intake. Durstig-Gal4-driven GFP revealed that a complex pattern of neurons is labeled
in the brain (Figure 1—figure supplement 1E). Because of this, we sought to isolate the individual
neurons responsible for water seeking.
Pattern refinement reveals a small group of neurons that promote
thirsty water seeking
We used genetic intersectional techniques to isolate the water seeking neurons embedded in the
Durstig pattern. First, subtracting R65D05 neurons from Durstig reduced activation-dependent
water seeking, indicating that the water seeking neurons in Durstig and R65D05 are the same
(Figure 2A). Activation of central brain R65D05 neurons promoted water seeking, whereas their
inactivation decreased seeking, indicating that central brain neurons in the R65D05 pattern (and not
ventral nervous system neurons or other non-neuronal cells) are both necessary and sufficient for
water seeking (Figure 2B,C; Figure 2—figure supplement 1A–E). Feeding behavior toward dry
sucrose was increased with R65D05 inactivation, as expected from previous reports (Figure 2—fig-
ure supplement 1F; Chen et al., 2016; Hentze et al., 2015; Hergarden et al., 2012). Second, we
expressed the GAL4 inhibitor GAL80 in various neurotransmitter classes of neurons and assessed
Durstig activation-driven water seeking. Subtracting out neurons with VGlut-Gal80 (vesicular gluta-
mate transporter enhancer) specifically suppressed Durstig water seeking (Figure 2—figure supple-
ment 2A,B). Next, we activated neurons captured by enhancer-Gal4 transgenes created with
Landayan et al. eLife 2021;10:e66286. DOI: https://doi.org/10.7554/eLife.66286 4 of 19
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A Durstig R65D05 B C
R65D05 **
1 1 * 1
Fraction on H2O
Fraction on H2O
Fraction on H2O
+otd-nlsFLPo
** X
0.5 ** 0.5 0.5
**
0 0 0
Durstig-Gal4 • • • • • R65D05-Gal4 • • • R65D05-Gal4 • • •
UAS-TrpA1 • • • • • UAS-STOP-TrpA1 • • • UAS-STOP-Shi • • •
R65D05-LexA • • • • otd-nlsFLPo • • • otd-nlsFLPo • • •
LexAOP-Gal80 • • • •
H2O Replete, Grid H2O Replete, Grid Thirsty, Grid
D Durstig R52A01 E F
** **
1 1
** 1
Fraction on H2O
Fraction on H2O
Fraction on H2O
0.5 0.5 0.5
0 0 0
Durstig-Gal4 • • • • • R52A01-Gal4 • • R52A01-Gal4 • •
UAS-TrpA1 • • UAS-TrpA1 • • UAS-Shi • •
R52A01-LexA • • •
LexAOP-Gal80 • • •
H2O Replete, Grid H2O Replete, Grid Thirsty, Grid
G H I SMP
SMP
R52A01 R65D05 CRE
Janu>
AD DBD CRE SytGFP
DenMark
SEZ SEZ
Janu-spGal4
Janu>myrGFP Brp
J 1
K *
1
**
Fraction on H2O
Fraction on H2O
*
0.5 0.5
0 0
R52A01-AD • • • • R52A01-AD • • • •
R65D05-DBD • • • • R65D05-DBD • • • •
UAS-TrpA1 • • • • • • UAS-Shi • • • • • •
Grid Open Grid Open
H2O Replete Thirsty
Figure 2. Isolation of Janu thirst neurons from the Durstig pattern. (A) Subtraction of R65D05 neurons
(R65D05>Gal80) from the Durstig pattern decreases Durstig activation water seeking. One-way ANOVA/Dunnett’s
compared to Durstig>TrpA1. (B) Activation of R65D05 neurons specifically in the central brain promotes water
seeking. otd-nlsFLPo expresses Flippase in the central brain and not the ventral nervous system, to remove the
Figure 2 continued on next page
Landayan et al. eLife 2021;10:e66286. DOI: https://doi.org/10.7554/eLife.66286 5 of 19
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Figure 2 continued
STOP cassette from UAS-STOP-TrpA1, thus restricting TrpA1 expression to central brain R65D05 neurons. (C)
Inactivation of R65D05 neurons specifically in the central brain inhibits thirsty water seeking. (D) Subtraction of
R52A01 neurons (R52A01>Gal80) from the Durstig pattern decreases Durstig activation (Durstig>TrpA1) water
seeking. One-way ANOVA/Dunnett’s compared to Durstig>TrpA1. (E) R52A01 neuron activation increases water
seeking. (F) R52A01 inactivation decreases thirsty water seeking. (G) A split-Gal4 (spGal4) with R52A01-AD and
R65D05-DBD forms functional GAL4 (Janu-spGal4) selectively in the R52A01/R65D05 overlap. (H) Expression
pattern of Janu-spGal4 in the brain, counterstained with the Brp synaptic marker. (I) Janu neuron polarity, revealed
by presynaptic synaptotagmin-GFP (UAS-sytGFP, green) and dendrite localized UAS-DenMark (magenta). (J) Janu
neuron activation increases water seeking. (K) Janu neuron inactivation decreases thirsty water seeking. Statistics
are one-way ANOVA/Tukey’s or Kruskal-Wallis/Dunn’s, unless indicated otherwise.
The online version of this article includes the following figure supplement(s) for figure 2:
Figure supplement 1. Characterization of R65D05-Gal4 neurons for thirst and hunger behaviors.
Figure supplement 2. Characterization of R52A01-Gal4 neuron for thirst behaviors.
Figure supplement 3. Janu neuron water seeking and feeding behavior characterization.
Figure supplement 4. Anatomy of individual Janu neurons.
enhancer fragments taken from the VGlut locus, and we identified R52A01-Gal4 neurons as being
able to promote water seeking (Figure 2—figure supplement 2C,D). Subtracting R52A01 neurons
from the Durstig pattern blocked Durstig activation from increasing water seeking, indicating that
Durstig water seeking neurons were also found in R52A01 (Figure 2D). Like R65D05, R52A01 activa-
tion increased and R52A01 inhibition decreased water seeking (Figure 2E,F). Subtracting out neu-
rons with VGlut-Gal80 decreased thirsty water seeking in both TrpA1-activated R52A01 and
R65D05, suggesting that the water seeking neurons were shared between these two Gal4 driver
transgenes (Figure 2—figure supplement 2E,F). To test if R65D05 and R52A01 labeled the same
water-seeking neurons, we assembled a split-Gal4 (spGal4) derived from these two enhancer frag-
ments that we named Janu (the Estonian for thirst) (Figure 2G). Janu-spGal4 is R52A01-Gal4.AD;
R65D05-Gal4.DBD. Janu-spGal4 is expressed in four bilaterally symmetric neurons in the central
brain (Figure 2H). Janu neuron postsynaptic inputs are confined to the subesophageal zone (SEZ),
and their presynaptic outputs are distributed between the SEZ, the crepine region (CRE), and the
superior medial protocerebrum (SMP) (Figure 2I). TrpA1 activation of Janu neurons increased water
seeking, albeit to a lesser extent than either parental Gal4 driver alone (Figure 2J). Conversely,
acute inactivation of Janu neurons decreased thirsty water seeking (Figure 2K). The behavioral
effects of both Janu activation and inactivation on water seeking mapped to the central brain, similar
to the parental enhancer-Gal4s for Janu-spGal4 (Figure 2—figure supplement 3A–C). Interestingly,
feeding behavior was increased when Janu neurons were acutely inactivated, the opposite effect as
compared to water seeking (Figure 2—figure supplement 3D,E). Thus, the Janu neurons promote
water seeking and inhibit feeding behavior.
GABAergic local interneurons in the SEZ promote water seeking
To identify the specific neurons that control water seeking and feeding behavior, we characterized
the individual Janu neurons. First, we used a stochastic labeling technique to determine the anatomy
of each Janu neuron (Figure 2—figure supplement 4). Two of the four central brain Janu neurons
project locally in the SEZ, and the other two neurons project from the SEZ to the CRE and/or the
SMP. Second, we determined Janu neurotransmitter/neuromodulator identity using immunohis-
tochemistry with antibodies for specific neurotransmitter classes of neurons and for neuropeptides.
We found that the two local SEZ neurons were GABAergic, expressing the VGAT vesicular trans-
porter for GABA; we named them Janu-GABA1 and Janu-GABA2 (Figure 3A–H). VGAT precisely
localized with nearly all Janu-GABA axonal varicosities, and did not localize with other Janu neurons.
Knockdown of VGAT with either of two distinct RNAi transgenes in the Janu neurons decreased
water seeking in thirsty animals (Figure 3I; Figure 3—figure supplement 1A). Knockdown of Gad1,
encoding the GABA biosynthetic enzyme Glutamic acid decarboxylase 1, in Janu neurons also
decreased thirsty water seeking (Figure 3J). To ask if Janu-GABA neurons were responsible for pro-
moting water seeking when Janu neurons were activated in water replete flies, we knocked down
Gad1 while simultaneously activating the Janu neurons with TrpA1. This manipulation blocked
Landayan et al. eLife 2021;10:e66286. DOI: https://doi.org/10.7554/eLife.66286 6 of 19
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A B C D
SEZ
Janu-GABA1
Ja FLAG VGAT FLAG VGAT
E F G H
SEZ
Janu-GABA2 FLAG VGAT FLAG VGAT
I H2O seeking J H2O seeking K H2O seeking
**
1 * 1 1
*
Fraction on H2O
Fraction on H2O
Fraction on H2O
*
0.5 0.5 0.5
0 0 0
R52A01-AD • • R52A01-AD • • • • R65D05-Gal4 • • •
R65D05-DBD • • R65D05-DBD • • • • UAS-TrpA1 • • •
UAS-VGAT.IR • • • UAS-Gad1.IR • • • • • UAS-Gad1.IR • •
Thirsty Thirsty Thirsty Replete
Grid Grid Open Grid
L H2O intake M H2O intake N Feeding behavior
100 80 1
H2O consumption, s
**
First intake bout, s
80
dry sucrose
Fraction on
60
60
40 0.5
40
20 20
0 0 0
R52A01-AD • • R52A01-AD • • R52A01-AD • • • •
R65D05-DBD • • R65D05-DBD • • R65D05-DBD • • • •
UAS-Gad1.IR • • UAS-Gad1.IR • • UAS-Gad1.IR • • • • •
Thirsty Thirsty Fed Hungry
Figure 3. Janu-GABA SEZ local interneurons specifically promote water-seeking behaviors. (A–D) Janu-GABA1 interneuron morphology in the SEZ of a
Janu>multicolor flip-out brain, compressed z-stack. Arrowhead in (A) points to the cell body. Boxed area is enlarged in (B–D), showing colocalization of
the Janu-GABA1 neuron (anti-FLAG) with immunoreactivity for VGAT (arrowheads in B–D). Single 1 mm confocal section. (E–H) Janu-GABA2 interneuron
morphology in the SEZ of a Janu>multicolor flip-out brain, compressed z-stack. Arrowhead in (E) points to the cell body. Boxed area is enlarged in (F–
Figure 3 continued on next page
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Figure 3 continued
H), showing colocalization of the Janu-GABA2 neuron (anti-FLAG) with immunoreactivity for VGAT (arrowheads in F–H). Single 1 mm confocal section. (I)
VGAT knockdown in Janu neurons decreases thirsty water seeking. (J) Gad1 knockdown in Janu neurons decreases thirsty water seeking. (K)
Simultaneous TrpA1 neuronal activation and Gad1 knockdown in R65D05 neurons blocked R65D05>TrpA1 increased water seeking in replete flies. (L)
No effect on water consumption time with Gad1 knockdown in Janu neurons in thirsty flies. (M) Decreased time to termination of first drinking bout
with Gad1 knockdown in Janu neurons in thirsty flies. (N) No effect on feeding behavior with Gad1 knockdown in Janu neurons. Statistics are one-way
ANOVA/Tukey’s or Kruskal-Wallis/Dunn’s.
The online version of this article includes the following figure supplement(s) for figure 3:
Figure supplement 1. Additional characterization of Janu-GABA neuron function in thirst behaviors.
activation-dependent water seeking (Figure 3K). Thus, the Janu-GABA neurons promote thirsty
water seeking and are capable of driving water seeking in water replete animals. To ask if Janu-
GABA neurons are specific to the seeking phase of thirst or if they drive a sequence of thirst behav-
iors, we tested water intake. We presented thirsty flies with a drop of water and measured total
water consumption time, the length of the first intake bout, and pharyngeal pumping rate. Gad1
knockdown in Janu neurons had no effect on total water consumption time, but it reduced the
length of the first intake bout (Figure 3L,M). Pharyngeal pumping rate was unchanged in thirsty
Janu neuron Gad1 knockdown flies, indicating that total intake and motor behaviors were unaffected
(Figure 3—figure supplement 1B,C). Finally, feeding behavior was unaltered in the Janu Gad1
knockdown flies (Figure 3N). Thus, the Janu-GABA neurons are thirst-driven water seeking inhibitory
local interneurons in the SEZ.
AstA neurons reciprocally regulate water seeking and feeding behavior
The Janu-AstA neuron is a bilaterally symmetric interneuron that projects ipsilaterally from the SEZ
to the ventral medial SMP, and that expresses the neuropeptide AstA (Figure 4A–G; Figure 4—fig-
ure supplement 1A, B). AstA knockdown in Janu-spGal4 neurons with either of two independent
RNAi transgenes reduced thirsty water seeking (Figure 4H,I; Figure 4—figure supplement 1C–E).
Moreover, loss-of-function AstA mutant flies showed markedly reduced water seeking, both as a
homozygote and when in trans to a small chromosomal deletion at the AstA locus (Figure 4J,K). To
ask if Janu-AstA neurons were responsible for promoting water seeking when Janu neurons were
activated in water replete flies, we knocked down AstA while simultaneously activating the Janu neu-
rons with TrpA1. This manipulation blocked activation-dependent water seeking (Figure 4L; Fig-
ure 4—figure supplement 1F). Thus, like Janu-GABA neurons, the Janu-AstA neurons promote
thirsty water seeking and are capable of driving seeking in water replete flies. This finding suggests
that there may exist a functional relationship between the Janu-GABA and the Janu-AstA neurons.
However, there are key functional differences between the Janu-GABA and Janu-AstA neurons.
AstA knockdown in Janu neurons did not affect total water consumption time, the length of the first
intake bout, pharyngeal pumping rate, or intake measured in the open-field assay (Figure 4M,N;
Figure 4—figure supplement 1G–I). Thus, Janu-AstA neurons are required for thirsty water seeking,
and they promote seeking when activated in water replete flies, but they have no role in water
intake. Interestingly, AstA in the Janu neurons inhibits feeding behavior in both well-fed and hungry
flies (Figure 4O; Figure 4—figure supplement 1J). Thus, the Janu-AstA neurons oppositely regulate
water seeking and feeding behavior. This is in contrast to the Janu-GABA neurons that are specific
to water-seeking behavior.
Neural activity and motivational properties of Janu neurons
To test if thirst and hunger regulate the activity of the Janu neurons, we used the TRIC genetic cal-
cium indicator that accumulates GFP in active neurons (Gao et al., 2015). The Janu-GABA1 neurons
were selectively activated in thirsty flies; we detected no thirst-dependent activity in any other Janu
neurons (Figure 5A,B). Moreover, hunger did not change the activity of any Janu neuron
(Figure 5C). These data indicate that the Janu-GABA1 neurons selectively increase activity with
increasing water deprivation.
Finally, we asked if Janu neuron activity imparts appetitive or aversive value to the fly. We mea-
sured positional preference in an open-field arena where the light-gated ion channel Chrimson,
Landayan et al. eLife 2021;10:e66286. DOI: https://doi.org/10.7554/eLife.66286 8 of 19
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A B E
SMP
Janu
AstA Janu AstA
C F
Janu Janu
D G
SEZ
AstA AstA
H H2O seeking I H2O seeking J H2O seeking K H2O seeking
** *
1 1 1 ** 1
Fraction on H2O
*
Fraction on H2O
Fraction on H2O
Fraction on H2O
0.5 0.5 0.5 0.5
0 0 0 0
R52A01-AD • • • Thirsty Replete
ici 1
R52A01-AD • • • co cy
l
/d / sk
ro
en
R65D05-DBD • • • nt
co/ sk1
co/ sk1
• • •
k1 k1
R65D05-DBD
tA s ol
l
ro
tA s stA s
r
nt
nt
• • •
ef
UAS-AstA.IR
1
k1
UAS-AstA.IR • • •
k
tA s
As A
UAS-Dcr2 • • • • UAS-Dcr2 • • • •
As
As
Thirsty, Grid Thirsty, Open Thirsty, Grid Open
L H2O seeking M H2O intake N H2O intake O Feeding behavior
1 100 80 1
H2O consumption, s
**
First intake bout, s
Fraction on H2O
80 **
dry sucrose
Fraction on
60
0.5
* 60
0.5
40
40
20 20
0 0 0 0
R52A01-AD • • • R52A01-AD • • R52A01-AD • • R52A01-AD • • • •
R65D05-DBD • • • R65D05-DBD • • R65D05-DBD • • R65D05-DBD • • • •
UAS-TrpA1 • • UAS-AstA.IR • • UAS-AstA.IR • • UAS-AstA.IR • • • • • •
UAS-AstA.IR • UAS-Dcr2 • • UAS-Dcr2 • • UAS-Dcr2 • • • • • •
Replete, grid Thirsty Thirsty Fed Hungry
Figure 4. The Janu-AstA neuron reciprocally regulates thirst and feeding behaviors. (A–G) Janu-AstA neuron morphology and AstA expression. (A)
Janu-AstA neuron morphology obtained from a multicolor flip-out brain. Green: anti-FLAG; magenta: anti-VGAT. Arrowhead points to cell body.
Process at bottom left is from a co-labeled Janu-GABA neuron. (B–D) The Janu-AstA neuron cell body is immunoreactive for AstA. Arrowheads points
to the cell body. (E–G) Colocalization of AstA immunoreactivity with Janu-AstA presynaptic endings in the medial SMP. Arrowheads point to a subset of
overlapping expression. Single 1 mm confocal section. (H, I) AstA knockdown in Janu neurons reduced thirsty water seeking and open water occupancy.
Figure 4 continued on next page
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Tools and resources Genetics and Genomics Neuroscience
Figure 4 continued
(J, K) Flies lacking AstA showed reduced thirsty water seeking and open water occupancy. (K) t-test. (L) Simultaneous TrpA1 neuronal activation and
AstA knockdown in Janu neurons blocked increased water seeking in replete flies. (M) No effect on water consumption time with AstA knockdown in
Janu neurons in thirsty flies. (N) No effect on time to termination of first drinking bout with AstA knockdown in Janu neurons in thirsty flies. (O) AstA
knockdown in Janu neurons increased food occupancy in well-fed and hungry flies. Statistics are one-way ANOVA/Tukey’s or Kruskal-Wallis/Dunn’s,
unless indicated otherwise.
The online version of this article includes the following figure supplement(s) for figure 4:
Figure supplement 1. Additional characterization of Janu-AstA neurons.
A Replete Thirsty
GABA2 D LED
GABA2 1
GABA1
Positional preference
GABA1 on
B 2 **
Relative Intensity
0
Replete
Thirsty
1 off
-1
0’ 5 10 15
0 Janu>Chrimson, ATR+
GABA1 GABA2 AstA Contra Janu>+, ATR+
Janu neuron
C 2 E 1
Relative Intensity
**
Fed Activation effect 0.5
Hungry
1
0
0 -0.5
GABA1 GABA2 AstA Contra Janu • • •
Janu neuron Chrimson • •
ATR • •
Figure 5. Thirst regulation of Janu neuron activity, and positive valuation of optogenetic activation of Janu. (A)
Examples of TRIC detection of neuronal activity (green) and the pattern of cells expressing the calcium indicator
(magenta) under water replete and thirsty conditions in Janu neurons, detected with anti-GFP (green) and dsRed
(magenta) antibodies. (B, C) Relative intensity of TRIC signal (green in A) to pattern (magenta in A) for the
individual Janu neurons in replete and thirsty (B), and well-fed and hungry (C) flies. Multiple adjusted t-test, with
FDR = 1%. (D) Positional preference of Janu>Chrimson (red) and Janu>+ (blue) water replete and well-fed flies
that were fed all-trans retinal (ATR). Positive values indicate attraction to LED-ON half of the rectangular arena,
controlled for arena side bias. (E) Quantification of D. Activation effect is the average positional preference for a 1
min bin at 4 min (activation lights off) subtracted from 9 min (activating LEDs on) (see also Figure 5—figure
supplement 1A). One-way ANOVA/Tukey’s.
The online version of this article includes the following figure supplement(s) for figure 5:
Figure supplement 1. Flies prefer activation of water seeking neurons.
Landayan et al. eLife 2021;10:e66286. DOI: https://doi.org/10.7554/eLife.66286 10 of 19Tools and resources Genetics and Genomics Neuroscience
expressed in Janu neurons, could be selectively activated in either half of an open-field arena.
Janu>Chrimson flies that were well fed and water replete were attracted to the activation side of
the arena (Figure 5D,E; Figure 5—figure supplement 1A). Flies showed a similar preference for
Chrimson activation of the R65D05 pattern that also contains the Janu water seeking neurons (Fig-
ure 5—figure supplement 1B,C). These findings suggest that water seeking driven by Janu neurons
is rewarding. Moving up a humidity gradient when thirsty may imply impending repletion to the fly,
and an end to the negative valence state of thirst (Betley et al., 2015).
Janu-AstA neurons are anatomically and functionally upstream of AstA-
R2-expressing NPF neurons
NPF promotes feeding behaviors in Drosophila (Shao et al., 2017; Shohat-Ophir et al., 2012;
Wu et al., 2003). Janu-AstA projects to the SMP, where NPF neurons elaborate synaptic endings.
Janu-AstA presynaptic outputs partially overlapped with NPF in the SMP (Figure 6A,B). Moreover,
synaptic GRASP (GFP Reconstitution Across Synaptic Partners) revealed that Janu-AstA neurons are
presynaptic to NPF neurons (Figure 6C; Macpherson et al., 2015). To test for signaling to NPF neu-
rons by AstA, we reduced expression of the two AstA receptors AstA-R1 and AstA-R2 in NPF neu-
rons using previously characterized RNAi transgenes (Yamagata et al., 2016). AstA-R2, but not
AstA-R1, knockdown decreased thirsty water seeking, and also increased feeding behavior, similar
to AstA function in the Janu-AstA neurons (Figure 6D; Figure 6—figure supplement 1A). AstA-R2
knockdown in NPF neurons, however, reduced both total water consumption time and the duration
of the first intake bout, indicating that AstA signaling to NPF neurons affects both water seeking
and water ingestion (Figure 6E,F; Figure 6—figure supplement 1B). Consistent with AstA-R2 func-
tioning as an inhibitory receptor in NPF neurons, we observed increased water seeking in flies
completely lacking NPF and in flies with NPF knocked down in NPF neurons (Figure 6G,H). Mea-
surement of neuronal activity in NPF neurons indicated that only two NPF neurons, the P1 neurons
and the L1-L neurons, showed any activity, and they both increased activity in thirsty flies (Figure 6I;
Figure 6—figure supplement 1C). These findings indicate that NPF neurons respond to thirst and
that NPF neurons receive AstA signals via AstA-R2 to reciprocally regulate thirst and hunger behav-
iors. Figure 7 summarizes our current understanding of the thirst circuitry and its relation to feeding
behavior.
Discussion
Thirst is an internal state encoded by discrete neural circuits that generate innate goal-directed
behavioral actions. We used an unbiased screen followed by genetic techniques to discover central
brain interneurons that regulate specific aspects of thirsty water seeking, and that contribute to a
specific motivational state. While sensory and interoceptive inputs are known, there is little under-
standing of how the brain forms the representation of thirst and coordinates sequences of behaviors
to achieve water repletion. The Janu-GABA water seeking and Janu-AstA to NPF water seeking and
feeding behavior circuit elements define specific functional steps of thirst representation, and they
provide a means to help understand the interactions of different need states.
The Janu-GABA neurons are GABAergic local interneurons whose activity is increased by thirst
and that promote thirsty water seeking. GABA in Janu-GABA is sufficient for driving water seeking
in the water replete state, and it is necessary for thirsty seeking. The role of the Janu-GABA neurons
appears to be limited to the seeking step of thirst behavior, since thirsty water consumption is
largely unaffected by their inhibition. The specific role for Janu-GABA in promoting persistence dur-
ing the first thirsty intake bout may reflect the end of the seeking component of the sequence of
thirst behaviors that may overlap with the beginning of the intake phase. Moreover, the Janu-GABA
neurons did not affect feeding behavior and are not activated by hunger. Thus, the Janu-GABA neu-
rons increase thirsty seeking to sources of water. The Janu-GABA neurons are functionally distinct
from the Fdg local interneurons that are also located in the SEZ (Flood et al., 2013; Pool et al.,
2014). The Fdg neurons increase intake of liquid food and promote proboscis extension, behavioral
functions we did not observe for the Janu-GABA neurons. We showed that the Fdg neurons may
also promote water seeking. However, we did not identify the specific neurons in the NP883-Gal4
pattern that are responsible for water seeking. Defining the source of the signal to drive Janu-GABA
dependent thirsty seeking and the mechanism of thirsty seeking require further elaboration of the
Landayan et al. eLife 2021;10:e66286. DOI: https://doi.org/10.7554/eLife.66286 11 of 19Tools and resources Genetics and Genomics Neuroscience
A B
NPF Janu>myrGFP Janu>myrGFP NPF
C syb:GRASP AstA AstA syb:GRASP
D H2O seeking Feeding behavior E H2O intake F H2O intake
1 ** 100 * 80
H2O consumption, s
*
Fraction on source
**
First intake bout, s
* 80 60
* 60
0.5 40
40
20 20
0 0 0
NPF-Gal4 • • • • • • • • NPF-Gal4 • • • •
UAS-AstA-R2.IR • • • • • • • •
UAS-AstA-R2.IR • • • •
H2O status Thirsty Thirsty Replete Replete
Thirsty Thirsty
Food status Fed Fed Hungry Fed
Source H2O, Grid H2O, Open Dry Sucrose Dry Sucrose
G H2O seeking H H2O seeking I Neuron activity
1 1
*** Replete
Fraction on H2O
Fraction on H2O
Relative Intensity
* * 2 Thirsty
**
0.5 0.5
1 *
0 0 0
NPF-Gal4 • • P1 L1-l
2
ici l
co cy
l
sk
tro
ro
en
UAS-NPF.IR • •
nt
NPF neuron
2 on
PF
c
/N
H2O status Thirsty
ef
2
F sk
/d
F sk
Food status Fed
NP
NP
Source H2O, Grid
4h thirsty, Grid 8h thirsty, Grid
Figure 6. NPF neurons are postsynaptic to Janu-AstA neurons via the AstA-R2 receptor, and NPF inhibits water seeking. (A) Janu-AstA presynaptic
endings in boxed area imaged for immunohistochemistry. (B) Janu-AstA presynaptic endings partially overlap with NPF immunolabeling. Single 1 mm
confocal section. Yellow arrowheads point to overlap. (C) Synaptic GRASP (syb:GRASP) between R65D05-Gal4>UAS-syb:spGFP1–10 and NPF-
LexA>LexAOP-spGFP11 in thirsty flies. Single 1 mm confocal section. Yellow arrowheads point to GRASP GFP signal that overlaps with AstA
Figure 6 continued on next page
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Figure 6 continued
immunoreactivity in the superior medial protocerebrum. (D) Behavioral effects of AstA-R2 RNAi in NPF neurons. (E) AstA-R2 knockdown in NPF neurons
decreased total water consumption time. (F) AstA-R2 knockdown in NPF neurons decreased the length of the first water intake bout. (G) Flies mutant
for NPF show increased water seeking when mildly water deprived (4 hr). Mann-Whitney t-test. (H) NPF knockdown in NPF neurons increased water
seeking in mildly water deprived flies (8 hr). (I) NPF neuron activity in thirsty flies with TRIC. Activity was detected only in P1 and L1-l NPF neurons. t-test.
Statistics are one-way ANOVA/Tukey’s or Kruskal/Wallis/Dunn’s, unless indicated otherwise.
The online version of this article includes the following figure supplement(s) for figure 6:
Figure supplement 1. Additional characterization of the role of NPF neurons in thirst behaviors.
circuitry for thirst and other behaviors in the SEZ region of the Drosophila brain. We speculate that
the Janu-GABA neurons may function upstream or downstream of the osmosensory ISN neurons
that, when inhibited, promote water intake (Jourjine et al., 2016). Janu-GABA neuron activation in
the SEZ is predicted to lead to inhibition of SEZ neurons, perhaps to repress competing behavioral
states represented in the SEZ. Alternatively, the Janu-GABA neurons may repress repressor neurons
of thirsty seeking, that remain to be identified.
Janu-AstA is a bilaterally symmetric AstAergic projection interneuron with inputs in the SEZ and
outputs in the medial SMP. AstA in the Janu-AstA neuron promotes thirsty water seeking, and it
inhibits feeding behavior in a hunger-independent manner. For thirst, AstA in the Janu-AstA neuron
was specific to thirsty seeking with no impact on water intake, indicating a functional role similar to
Janu-GABA for thirst. However, we did not detect changes in Janu-AstA neuronal activity with either
thirst or hunger. This lack of response may indicate a tonic role for AstA in feeding and thirst. Tonic
release of neuropeptides can tune neural circuit function, including the peptidergic modulation of
hunger (Fu et al., 2004; Tu et al., 2005). Peptidergic signaling can also be tuned by changing the
amount of the receptor in the receiving neuron, as was demonstrated for sensory tuning by the
short-NPF (sNPF) neuropeptide (Root et al., 2011). Interestingly, knockdown of either AstA or
GABA in the Janu neurons blocked water seeking evoked by Janu activation. Janu-GABA and Janu-
AstA may work in series in the same circuit, or in parallel in separate circuits that converge, to con-
trol thirsty-seeking behaviors.
Whereas Janu-GABA and Janu-AstA may work together for thirsty seeking, Janu-AstA also regu-
lates feeding behavior, indicating branched functionality in water seeking circuitry. Janu-AstA trans-
mits thirst and hunger information to NPF neurons through the AstA-R2 receptor on NPF neurons,
where NPF coordinately regulates thirsty seeking and feeding behavior. NPF is well studied in Dro-
sophila and in vertebrates as a promoter of feeding behavior – our work indicates a reciprocal role
A high hemolymph B
osmolarity
off Thirst
ISN Hunger Behavior Measure Janu-GABA Janu-AstA NPF
hunger
Water seeking Hygrotaxis Promotes Promotes Inhibits
on First bout length Promotes – Inhibits
Janu- Janu-
AstA GABA
Water ingestion Intake time – – Inhibits
AstA-R2 AstA-R2
Pumping rate – – –
NPF NPF
water Feeding Foraging – Inhibits Promotes
seeking
water feeding
intake behavior
Figure 7. Thirst interneuron circuitry in Drosophila. (A) Functional relationship for thirst and feeding behavior. The dotted lines represent putative
connectivity between interoceptive sensory neurons (ISN) and targets neuropeptide F (NPF) and Janu neurons. (B) Functions of GABA, AstA, and NPF
in their respective thirst interneurons.
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for NPF in thirst behavior (Chen et al., 2019; Luquet et al., 2005; Pool and Scott, 2014; Shen and
Cai, 2001). Coordinated regulation of thirst and hunger is most clearly manifested behaviorally as
prandial thirst (thirst induced by feeding) and as dehydration anorexia (thirst suppression of feeding)
(Fitzsimons, 1972; Zimmerman et al., 2017). Janu-AstA and AstA-R2-positive NPF neurons join a
small list of neurons that oppositely regulate thirst and hunger behaviors: the osmosensory ISN neu-
rons oppositely regulate food and water intake, and Ion Transport Peptide (ITP) neurons promote
thirst and inhibit food intake (Gáliková et al., 2018; Pool et al., 2014). It remains unclear if all these
neurons support water seeking, or if they function specifically during ingestion.
Interestingly, two NPF neurons, the P1 and L1-l neurons, showed increased activity in thirsty flies.
Hunger also selectively increases the activity of these two NPF neurons (Beshel and Zhong, 2013).
This might indicate that the P1 and L1-l represent an internal state imbalance rather than a specific
internal state like hunger or thirst. Additionally, the direction of their regulation – increased activity
when thirsty – is opposite of what is expected for NPF in these neurons to inhibit thirsty seeking.
Thus, we suggest that Janu-AstA may synapse onto different NPF neurons that, like Janu-AstA, do
not exhibit state-dependent changes in neuronal activity, but that promote thirst and inhibits feed-
ing behavior. A potential mechanism may be through NPF-NPF neuronal synapses: both the P1 and
the L1-l neurons express the NPF receptor NPFR (Shao et al., 2017). Moreover, it is likely that one
or more of the water-seeking circuit elements we discovered feeds into navigational decision making
circuitry to direct hygrotaxis toward a water source and to suppress other goal directed behavior in
a dynamic fashion (Hulse et al., 2020; Lyu et al., 2020).
Optogenetic activation of the Janu or the R65D05 neurons induces place preference, indicating
that animals ‘like’ activation of these neurons. Thirst and hunger are deprivation states that have a
negative valence, therefore the Janu neurons likely do not encode the state of deprivation
(Burnett et al., 2016; Leib et al., 2017; Sutton and Krashes, 2020). We propose that, instead, they
encode the anticipation of terminating a thirsty state. To a thirsty fly, the presence of a humidity gra-
dient likely predicts impending water intake and repletion, which may have rewarding value.
Materials and methods
Strains and culturing
All strains were outcrossed for five generations to the Berlin genetic background. Flies were raised
on standard food containing agar (1.2% w/v), cornmeal (6.75% w/v), molasses (9% v/v), and yeast
(1.7% w/v) at 25˚C and 60% humidity in a 16:8 light:dark cycle. For experiments with UAS-Shibirets
and UAS-TrpA1, flies were reared and held at 18–23˚C prior to testing. For split-Gal4, RNAi, and
UAS-STOP-effector/FLP experiments, adult F1 progeny were held at 29˚C for 2–3 days prior to test-
ing to enhance transgene activity. Supplementary file 1 provides a list of the InSITE-Gal4 strains
screened by neuronal activation for feeding behavior, and their seeking index (fraction on food,
InSITE-Gal4>TrpA1 – InSITE-Gal4>+) (Gohl et al., 2011). Durstig-Gal4 is an InSITE-Gal4 enhancer
trap inserted into the first intron of CG4502 on chromosome 2. All strains used in this study are
described in Supplementary file 2.
Behavioral measurements
Genotype and treatment were blinded for all behavioral counts. Groups of approximately 20 adult
males were collected 1–2 days prior to the experiment. One group is an n = 1. Matched experimen-
tal and control groups were tested within day, and experiments were performed across days, to
account for between-day variability. For water deprivation, flies were placed in empty culture vials
containing 1 mL 5% sucrose that was dried onto a 2.5 15 cm strip of Whatman 3 MM filter paper.
For food deprivation, flies were placed into empty culture vials containing water saturated 3 MM fil-
ter paper. Dry, rough cut cane sugar (La Perruche Pure Cane Rough Cut Cubes) was used in all food
seeking experiments, except for the original screen that used standard fly food. Sources of food or
water were presented to flies either on a small square of Parafilm or in the cap of a 0.65 mL micro-
centrifuge tube. Water was made inaccessible by gluing a 210–300 mm mesh grid onto a cap. Flies
were deprived of food or water for the times indicated in Supplementary file 3. Thin-walled Plexi-
glas behavioral chambers were designed with two side-by-side arenas, each arena measuring 45
7510 mm. Chambers were designed and built by IO Rodeo (Pasadena, CA); design files are
Landayan et al. eLife 2021;10:e66286. DOI: https://doi.org/10.7554/eLife.66286 14 of 19Tools and resources Genetics and Genomics Neuroscience
available from the company. The chambers and sources were acclimated to the testing temperature
in a Peltier incubator, and flies were acclimated into the chambers for 10 min prior to introducing
the source (IN45, Torrey Pines Scientific). For TrpA1 and Shibire thermogenetics, flies were tempera-
ture acclimated for 20 min before being introduced into the chamber. Humidity was measured using
an EK-H4 multiplexer with SHT71 sensors (Sensirion). The incubator maintained 30–40% relative
humidity. For thermogenetic activation experiments with UAS-STOP-TrpA1-mCherry, flies were pre-
acclimated for 20 min in food or deprivation vials, then 10 min in the behavioral arena, as previously
reported (Asahina et al., 2014). Flies were filmed from above with Logitech C510 web cameras at
10 fps with the arena placed on white light LED panel (Edmund Optics). The number of flies on the
source was divided by total number of flies to determine the fraction of flies on the source. In binary
choice experiments, dry sucrose and water were placed in closely apposed receptacles just prior to
performing the experiment.
Locomotor speed was calculated per group as the total distance traveled divided by the total
time each individual was tracked for 10 s intervals every 5 min, and then averaged across the 10 min
and 15 min time points, allowing for acclimation to the arena (Wolf et al., 2002).
To measure intake in the open-field arena, water was made to 0.2% w/v erioglaucine and pre-
sented to flies for 20 min in the behavioral chamber. Consumption was determined spectrophoto-
metrically in cleared fly extracts at 628 nm by a BioTek Epoch two microplate reader, background
subtracted with flies fed undyed water, and was normalized to the enhancer-Gal4>+ control strain.
Intake time and pumping rate were measured essentially as previously described (Manzo et al.,
2012). Flies were glued to slides with clear nail polish while cold anesthetized and allowed to
recover at 60% humidity and 25˚C for 1–2 hr. Consumption time was measured by repeatedly pre-
senting the proboscis with a water drop extruded from the end of a 5 mL calibrated micropipet
(Avantor), until flies failed to extend the proboscis to four sequential presentations. The first bout
length was the time from first engagement to first complete disengagement with the water drop.
Pumping rate was determined with manual counts of video recordings of pharyngeal contractions
during the initial 10 s of engagement, or shorter if the flies disengaged earlier.
We used a custom-built device for determining the positional preference of optogenetically stim-
ulated flies (Flidea). Groups of about 10 flies were kept in the dark on standard food containing 35
mM all-trans retinal for 2–4 days (Sigma). They were placed in an 80 40 3 mm arena with a clear
roof and white acrylic floor. Optogenetic LEDs (617 nm) were arrayed 1 cm below the arena and
were switched to allow illumination of either 40 40 mm side of the arena. Optogenetic stimulation
was at 40 Hz for 200 ms every second. Flies were allowed to acclimate to the arena, and then were
given a 5 min off, 5 min on, 5 min off pattern of stimulation, with the ON side randomly assigned.
Filming was done at 2 fps in dim ambient light. Object recognition and position in the arena was
determined using custom developed DTrack software (Ro et al., 2014). Fly positional preference
was calculated as the number of flies on a side divided by the total number of flies. Experiments
where flies showed a strong side bias prior to optogenetic stimulation were excluded from further
analysis. The resulting positional information was averaged into 20 s bins. The activation effect is the
positional preference averaged across minute nine minus across minute 4, such that a positive value
indicates increased occupancy of the LED-on side when the LEDs are on. The experiment was
repeated with flies from 5 to 8 separate crosses across different days.
Statistical measurements were made with Prism 8.0 (GraphPad). One-way ANOVA followed by
Tukey’s post-hoc comparisons (or Bonferroni post-hoc planned comparison) were used when data
did not show unequal variance by the Brown-Forsythe test, otherwise the Kruskal-Wallis test fol-
lowed with Dunn’s post-hoc was used. t-tests were two-tailed (Supplementary file 3). Error bars are
the SEM. Dots overlaid on the bar graphs are the value measured for individual groups. No statistical
methods were used to predetermine sample sizes. Sample sizes are similar to those used in other
Drosophila behavioral experimental paradigms, and they were held constant with rare exceptions.
No data was excluded using statistical methods.
Immunohistochemistry
Adult fly brains were dissected in PBS with 0.1% Triton X-100 (PBT), fixed overnight at 4˚C in PBT
with 2% paraformaldehyde, blocked in PBT with 5% normal goat serum and 0.5% bovine serum albu-
min, and immunostained as described previously (Kong et al., 2010). Antibodies were rabbit anti-
GFP (1:1000, Life Technologies), chicken anti-GFP (1:1000, AbCam), mouse anti-GFP for GRASP
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